This proposal establishes the need for a 3-laser 2-camera Amnis ImageStreamX Mark II imaging flow cytometer with 12 imaging channels for the University of North Carolina at Chapel Hill. Imaging cytometry provides high-speed multi-parametric analysis of complex populations of cells while also allowing the fine imagining of each single cell and cellular complexes. This provides a powerful visualization of cellular processes such as nuclear translocation or compartmental co-localization of multiple intracellular or membrane associated molecules. Traditional imaging cytometry may show cellular localization of 2-3 molecules simultaneously, but is limited in the numbers of cells that may be analyzed and the number of molecules that can be simultaneously measured. Photometric algorithms allow the calculation of nuclear translocation, co- localization, nuclear to cytoplasmic ratios, and powerful statistical analysis of the measured events. The combination of imaging and flow cytometer provides a unique tool for the study of cell biology toward the advancement of medicine. The ability to perform this high-content imaging provides justification for an additional laser and imaging channels. The ImageStream will be available to university faculty to advance medical research. The instrument will be housed in the UNC Flow Cytometry Core Facility and supported by the facility staff. The UNC Flow Cytometry Core Facility serves as a university-wide recharge core for flow cytometry. Imaging experts within the university and collaborating labs will provide the initial start-up expertise along with training by the manufacturer and online data analysis service. This will be the first instrument of its kind on the UNC campus. This application comes from 9 major and 6 minor users with active NIH research funding (Direct Costs) totaling $49,633,048. Supplemental funds for instrument staffing will be provided by the UNC School of Medicine for the first year. Additional funds will be provided by the School of Medicine for service and maintenance costs not recovered from the core recharge center. IMPACT. The impact of this instrument will be immediate, as evidenced by preliminary data provided in this application obtained from an Amnis FlowSight demonstration, current flow cytometry data lacking imaging capabilities and imaging data lacking high throughput analysis available only from the ImageStream. The 12- imaging channels will allow data acquisition from 3 laser lines and allow for future laser upgrades as demand indicates. Specific projects propose work to explore cellular processes in cancer, HIV infection, autoimmunity, environmental toxicology, diabetes, sickle cell disease, vascular remodeling, signal relay systems, innate immunity and neural differentiation. Through educational efforts led by the core facility staff, itis anticipated that many more investigators will exploit the versatile capabilities of this instrument and ideally promote new areas of scientific research.